ABSTRACT While multidrug-resistance transporters including P-gp and MRP2 are generally studied for their role in exporting drugs and foreign compounds from the cell, our studies indicate that these efflux pumps expressed at the apical surface of intestinal epithelial cells provide a critical link in communication between sentinel functions of mucosal barriers and the immune system. Understanding how this P-gp/eCB anti-inflammatory arm is regulated will provide crucial insight into how dysfunction may promote intestinal inflammation and help identify potential new therapeutic targets. Because the resident microbiota is known to contribute to tolerance and homeostasis in the healthy intestine, the central hypothesis we aim to test is whether the normal microbiota actively drives the P-gp/eCB axis to prevent unnecessary inflammation. Our pilot studies indicate that the microbiota does influence P-gp expression and function, providing a unique foundation for further cause-effect studies. No data have previously demonstrated a link between the microbiota and eCBs or any other epithelial lipid signals, and may well provide great insight into a novel system. Bridging this gap could help explain how commensal bacteria can stabilize a state of tolerance and how genetic modification of specific pathway elements might predispose individuals to conditions of inflammatory bowel disease (IBD). To begin addressing these questions, in Aim 1 of this application will combine in vitro (including human colonoids) and in vivo murine model systems, as well as use healthy and UC patient stool, to more deeply understand the microbial consortia that collectively maximize P-gp expression and function. Aim 2 is designed to identify the microbial metabolites that drive activation of P-gp expression and eCB secretion to maintain an anti-inflammatory tone in the intestinal epithelium. Thus, transcriptomics and metabolite analyses will be performed to provide new information regarding microbial genes, gene clusters, and their metabolic products implicated in maintaining an anti- inflammatory tone in the intestinal epithelium through regulation of the P-gp/eCB axis. In Aim 3 we will employ novel computational methods will to uncover the inter-microbial network responses and the ecological structure of a stable community that is able to induce P-gp expression. Collectively, knowledge of the pathways that coordinate the maintenance of the P-gp/eCB axis will require a comprehensive understanding of distinct signals regulating intestinal homeostasis, how multiple signals are integrated in the complex intestinal environment, and pathways that modulate host-microbe interactions. Consequently, this proposal will directly advance novel biological principles with guidance of new therapeutic intervention strategies.